Transgene assay using stable agrobacterium rhizogenes transformation

ABSTRACT

A novel method is described for the screening of gene elements of interest using hairy roots of chimeric plants transformed with  Agrobacterium rhizogenes.

This application is a divisional of U.S. application Ser. No. 09/386,605filed Aug. 31, 1999, now

U.S. Pat. No. 7,807,865, that claims priority from U.S. provisionalpatent application number 60/098,402, filed Aug. 31, 1998, each of thedisclosures of which are herein incorporated by reference in theirentirety.

FIELD OF THE INVENTION

The present invention relates in general to a new method of screeninggenetic elements of interest for functionality, and more particularly tosuch a method utilizing Agrobacterium rhizogenes to transform planttissue in a manner forming a chimeric plant expressing or containing thegenetic element of interest in transgenic root tissue.

BACKGROUND OF THE INVENTION

Agrobacterium rhizogenes is a soil bacteria that is known to infectwounded root tissue and that transfers a portion of its bacterialplasmid, the Ri plasmid, to the plant. The Ri-T-DNA that is transferredto the plant induces the formation of adventitious roots and thesegenetically transformed roots can be regenerated into whole plants thattransmit the Ri T-DNA to their progeny. Agrobacterium rhizogenes has,therefore, been used to generate stably transformed whole plants. In oneapplication of this technology, secondary metabolites can be producedfrom culture using this method. With the advent of genomics-baseddiscovery of genes and genetic elements, new methods are needed tofacilitate the rapid screening of the large numbers of genes (or geneticelements) that are becoming available. Typically, genes of interest arecloned and then stably transformed using Agrobacterium tumefaciensmediated delivery or by a particle gun method into plants for functionalanalysis of the gene or genetic element. This method can take up to 9months for transgenic plants, such as soybean, to be transformed andready for testing. This is a slow and inefficient process. Therefore,there is a need for a rapid method of screening large numbers of genesand gene constructs in planta for functionality.

SUMMARY OF THE INVENTION

The present invention relates to a rapid, in planta method for screeninga genetic element for functional activity. It has been discovered thatby utilizing Agrobacterium rhizogenes to transform plant tissue in amanner producing a chimeric plant having only transgenic root tissue,with the remainder of the plant being non-transgenic, transgenic tissuecontaining a selected genetic element can be available for testingwithout having to produce stably transformed whole plants. The methodgreatly reduces the time required to screen large numbers of geneticelements and permits functional testing in about 2 to 3 months from thestart of the transformation process.

Therefore, in one preferred embodiment, the present invention provides amethod for producing a stable chimeric plant having transgenic roottissue that comprises obtaining an explant, inoculating the explant withAgrobacterium rhizogenes containing an exogenous genetic element capableof being transferred to the explant, culturing the inoculated explant ina manner permitting transgenic root development, and producing a stablechimeric plant with transgenic root tissue. This transgenic root tissueis available for testing of the functionality of the genetic elementintroduced therein by standard methodology relevant to the geneticelement being tested.

Among the many aims and objectives of the present invention include theprovision of a method providing for an in planta assay for testing genesfor anti-pathogen or anti-insect activity; testing genes for enzymaticor metabolic activity; high-throughput gene trapping, promoter trapping,and enhancer trapping; optimizing constructs for gene expression andprotein production; testing constructs for gene expression beforesubmission for production of transgenic plants; and production of largeamounts of protein. Moreover, the present method provides a method ofproducing chimeric plants in soil, not in tissue culture, therebygreatly reducing the possibility of contamination and avoiding thedisadvantages associated with regenerating transgenic plants throughtissue culture methods.

Also provided are chimeric soybean plants produced by the methoddescribed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a representation of the plasmid map for pMON31873.

FIG. 2 is a representation of the plasmid map for pMON31892.

FIG. 3 is a representation of the plasmid map for pMON31896.

DETAILED DESCRIPTION OF THE INVENTION

In order to provide a clear and consistent understanding of thespecification and the claims, including the scope given to such terms,the following definitions are provided.

A “chimeric plant” is a plant with only a portion of its cellstransgenic. In the following examples the chimeric plants are defined ashaving transgenic roots but wild-type shoots, stems, and leaves.

A “genetic element of interest” can be a promoter, an intron, astructural gene, a fragment of a gene, a 3′ terminator, an enhancer, orany other genetic element that might affect gene expression, genefunctionality, or a combination thereof.

“Expression” means the combination of intracellular processes, includingtranscription and translation, undergone by a coding DNA molecule suchas a structural gene to produce a polypeptide.

“Promoter” means a recognition site on a DNA sequence or group of DNAsequences that provides an expression control element for a structuralgene and to which RNA polymerase specifically bind and initiates RNAsynthesis (transcription) of that gene.

“Regeneration” means the process of growing a plant from a plant cell(e.g., plant protoplast or explant).

“Structural gene” means a gene that is expressed to produce apolypeptide.

“Structural coding sequence” refers to a DNA sequence that encodes apeptide, polypeptide, or protein that is made by a cell followingtranscription of the structural coding sequence to messenger RNA (mRNA),followed by translation of the mRNA to the desired peptide, polypeptide,or protein product.

“Transformation” refers to a process of introducing an exogenous DNAsequence (e.g., a vector, a recombinant DNA molecule) into a cell orprotoplast in which that exogenous DNA is incorporated into a chromosomeor is capable of autonomous replication.

“Vector” means a DNA molecule capable of replication in a host cell orto which another DNA segment can be operatively linked so as to bringabout replication of the attached segment. A plasmid is an exemplaryvector.

“Exogenous” as used herein means any genetic element that is notnaturally occurring in a wild-type Agrobacterium rhizogenes organism

According to the present invention, there is provided a method for therapid in planta testing of an exogenous genetic element in a chimericplant. The plant is produced by transformation with Agrobacteriumrhizogenes. This process requires the use of a wild-type Agrobacteriumrhizogenes strain that transfers genes that encode for production ofplant growth regulators that stimulate hairy root formation to theinfected plant tissue during the transformation process. Numerousstrains of Agrobacterium rhizogenes are known, and any strain thatefficiently transforms the plant of interest may be used. It isunderstood, however, that some strains are more virulent than others andcertain strains may not be used with all plant species because of thelevel of virulence. Thus, the plant species being transformed and thestrain of Agrobacterium rhizogenes being used should be compatible. Mostpreferably, the highly virulent strain K599 is used with plant speciessuch as soybean and potato, but a less virulent strain may be needed fortomato.

In addition to the wild-type Agrobacterium rhizogenes strain that is tobe used for the transformation, a construct containing the geneticelement to be tested for functionality in planta is added to theAgrobacterium rhizogenes in the form of a binary plasmid, a piece ofcircular DNA. The plasmid may take many forms known in the art, buttypically requires an origin of replication that allows for stableplasmid retention in Agrobacterium rhizogenes; a suitable selectablemarker resistance gene that allows for selection of the plasmid inAgrobacterium rhizogenes; two DNA border sequences that determine thebeginning and end points of the DNA that is to be transferred to theplant cell; and a construct containing the genetic element to be testedthat is flanked by the before mentioned DNA border sequence. Theconstruct containing the genetic element of interest will typicallyinclude in linear sequence a promoter, promoter elements, a structuralgene, and a 3′ terminator, and the genetic element being tested may beany one of these elements.

Suitable selectable marker genes include, but are not limited to,antibiotic resistance markers such as the neomycin phosphotransferasegene, which confers resistance to kanamycin. Other preferred selectablemarkers are genes that confer tolerance to the glyphosate herbicide asdescribed in U.S. Pat. Nos. 5,463,175 and 5,633,435, herein incorporatedby reference.

If the genetic element being tested is a promoter sequence, theconstruct will require a reporter gene. Suitable reporter genes include,but are not limited to genes encoding for green fluorescent protein(GFP), β-glucuronidase (GUS), chloramphenicol acetyl transferase (CAT),and luciferase.

If the gene element of interest is a structural gene, the construct willrequire the elements needed for expression of the structural gene in aplant, including a promoter sequence and a 3′ non-translatedtermination/polyadenylation site and, optionally, an intron. Suitablepromoters include constitutive or root-specific promoters, such as, butnot limited to, enhanced 35S promoter from cauliflower mosaic virus(e35S CaMV), figwort mosaic virus promoter (FMV), the sugarcanebadnavirus promoter, the actin promoter from rice, the ubiquitinpromoter from maize, the nos promoter, the RB7 promoter, and the 4AS1promoter. Any suitable 3′ non-translated regions may be included in thevector containing the genetic element to be tested, including but notlimited to the 3′ region from the Agrobacterium tumor inducing (Ti)plasmid gene, such as the nopaline synthase gene (nos), and plant genessuch as the soybean 7s storage protein gene and pea ssRUBISCO E9 gene.Suitable introns are known in the art and may include the intron fromthe rice actin gene or an intron from a wheat heat shock protein.

Methods for constructing the vectors as described herein and means forintroducing such vectors into Agrobacterium rhizogenes are described inthe relevant literature, such as Sambrook et al. (Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1989) or Ausubel et al. (Current Protocols inMolecular Biology, John Wiley and Sons, New York, N.Y., 1995).

A structural gene being tested in the method of this invention may beany structural gene that might confer a beneficial trait to a plant,including but not limited to agronomic traits such as herbicidetolerance, yield improvements, insect or pathogen resistance, or qualitytraits such as enhanced or improved nutritional value, or otherproteins, enzymes or other biological product that may be produced in aplant.

Once the vector containing the genetic element to be tested isintroduced into the Agrobacterium rhizogenes, a suitable explant fromthe plant to be transformed is selected. The explant is derived from theplant of choice such that after inoculation with the vector containingAgrobacterium rhizogenes, the explant is capable of generatingtransgenic roots and maintaining a normal, non-transgenic stem, leavesand other plant structures. Preferably, the explant is a stem, hypocotylor other like structure. Most preferably, the explant is a hypocotylobtained by removing the roots from a growing cotyledon by cutting thehypocotyl about 2-3 cm below the cotyledonary leaves. It is alsopreferable to remove the plant tissue above the cotyledonary leaves aswell.

The explant is inoculated by contacting a cut or wounded portion of theexplant with a solution containing the Agrobacterium rhizogenes for aperiod of time suitable to permit transfer of the DNA to the explant.This typically occurs when the filter is dry. When the filter is airdried, it can take up to a week, but other methods of drying may also beused that would take less time. The Agrobacterium rhizogenes may becontacted by dipping the cut explant into the solution or vacuuminfiltration methods may be used. The bacterial solution may also beinjected into the explant by methods known in the art.

Chimeric plants are produced from transgenic roots after transformationwith Agrobacterium rhizogenes. Root growth can be initiated by placingthe inoculated end of the plant into liquid or solid media containingminimal salts media (i.e., ¼ strength Murashige and Skoog Salt Mixture(MS) [GibcoBRL, Cat. No 11117-074]). Hairy root formation can beobserved between two and three weeks after transformation withAgrobacterium rhizogenes. Once roots begin to grow, the entire plant maybe planted in soil or grown hydroponically. Generally, between 40 and90% of the hairy roots generated will be transformed with the geneelement of interest. All transgenic root growth is supported by theresources produced in the wild type shoots, stems, and leaves. Thismethod relies on the cotyledons or excised shoots to provide thenecessary resources for hairy root production, thus eliminating the needfor sugars or other carbon sources that would allow for easycontamination of the media. Production of hairy roots can be done in anon-sterile field or lab bench thus eliminating the need for sterilehoods and sterile lab equipment.

Once the transgenic roots are established, the genetic elementintroduced into the plant may be analyzed using any of the methodsfamiliar to those of skill in the art and appropriate for determiningthe functionality of the genetic element, including, but not limited to,immunochemical blots, Northern blots, Southern blots, extractions, plantpathogen assays, nodulation assays, enzyme assays, targeting assays,gene silencing assays, recombination assays, gene excision, functionalgenomics assay, PCR, and the like.

EXAMPLES

The following examples further illustrate the present invention. Theyare in no way to be construed as a limitation in scope and meaning ofthe claims.

Example 1 Transformation of Soybeans

Seed Sterilization

Petri dishes are filled with soybean seed and placed in a vacuumdesiccator. A beaker containing 200 mL of bleach and 2 mL ofconcentrated HCl is placed in the middle of a desiccator covered andvacuum applied. The vacuum is closed, and the seeds are allowed to sitfor 16 to 24 hrs.

Germinate Seeds

Pots are filled with silica sand and the sterilized soybean seed isplanted. The seed is germinated in a greenhouse for 7 days or untilfirst leaf expands. It is preferable that the soybean seeds are grown inthe greenhouse as this seems to improve the stability of the growinghypocotyl. First leaves are removed by cutting stem above cotyledons.The seedlings are transferred to a cold room at 4-6° C. (can be storedfor up to seven days).

Inoculation

Agrobacterium rhizogenes strain K599 containing the genetic element tobe tested is grown in LB media plus a plasmid selectable antibiotic in a30° C. shaker overnight. The cells are spun down by centrifugation(4,000×g, 10 min.) and resuspended in Agro resuspension solution ( 1/10strength B5 media plus 200 μM acetosyringone, 1 mM galacturonic acid,and 20 mM MES (pH5.4) to final OD_(600nm)=0.3). SORBAROD filters (IlaconLimited, type 7006, or Sigma, S6404, St. Louis, Mo.) that have beenplaced into a petri plate or microtiter plate are saturated with Agroresuspension solution. Remaining area of well or plate is filled withAgro resuspension solution (minus the Agro). Soybean hypocotyls are cutabout 2-3 cm below cotyledons and cut end of hypocotyls is placed intofilters and vacuum infiltrated for 5 minutes. The hypocotyls are placedin a growth chamber at 22° C., 18 hr. light/6 hr. dark photo-period andthe filters are permitted to dry until all of the Agro resuspensionsolution has evaporated and the filters have completely dried.

Root Initiation

There are two options (chambers or plates) for root initiation.

Chambers (1)

Find empty pipette tip boxes and remove lids. Sterilize in autoclave.Cover top with thin sheet of aluminum foil. Punch number of holes asneeded. Fill chamber with ¼ strength MS solution (pH 5.4) (optional isthe addition of low levels of selectable agents, i.e., kanamycin 50mg/L). Hypocotyls are removed from filters and placed in holes. Keepchambers in Percival at 22° C., 18 hr. light 6 hr. dark photo-period.

Plates (2)

Prepare ¼ strength MS (pH 5.4) plus 0.7% phytagel solution. Autoclave.Cool and pour into wide petri plates (optional is the addition of lowlevels of selectable agents, i.e., kanamycin 50 mg/L). Remove hypocotylsfrom filters and place in phytagel. Keep plates in growth chamber at 22°C., 18 hr. light/6 hr. dark photo-period.

Soybean Plantlet Culture

Remove any adventitious roots that may appear (these are roots above cutsite) until week three. If a large number of cotyledons appear to turnyellow, spray with fungicide by misting over top. Monitor water leveland replace as needed with ¼ strength MS (pH 5.4). This can be added tothe plates also. Do not let inoculated ends dry out. After three weeksroots should begin to grow from inoculated ends.

Hairy Roots

The number of transgenic hairy roots that form will be dependent on thecultivar. PI accessions tend to produce more hairy roots than cultivatedvarieties. On average between 5 and 10 independent transgenic roots canbe produced per hypocotyl.

Generally, between 40 and 70% of the hairy roots generated will betransformed with the genetic element introduced. In initiating roots inpresence of low levels of selectable agents (kanamycin 50 mg/L) up to90% of generated hairy roots will be transformed with the introducedgenetic element. When transgenes are linked to reporter genes (i.e.,GFP), transgenic roots can be selected based on expression of reportergenes.

Production of Chimeric Soybean Plants

Large amounts of hairy roots can be produced by planting the chimericplants in soil or grown hydroponically. The plant provides most of theenergy needed for hairy root growth. Only minimal salts are needed.Hairy root plants tend to be dwarf with early induced seed production.

The example below represents a study done on expression of ananti-fungal protein (AFP) from alfalfa (Alf). Two binary plasmids wereconstructed from pMON31873 (FIG. 1) using the Figwort Mosaic Virus (FMV)constitutive promoter, to drive expression of a cytoplasmically(pMON31892; FIG. 2) or extracellularly (pMON31896; FIG. 3) targetedAlf-AFP. Each construct was additionally linked to an enhanced 35Spromoter driving expression of the green fluorescence protein (GFP) andwas used to produce transgenic hairy-roots as described above. Fiveweeks after transformation, transgenic roots were individuallyharvested, analyzed for GFP expression by observing green fluorescenceunder a UV light and frozen. Protein was extracted from root-tissues andused in a standardized ELISA assay using an antibody made specificallyagainst Alf-AFP to determine the amount of Alf-AFP present. Table 1shows results of the study.

TABLE 1 Expression of Alf-AFP and GFP in hairy roots of soybeans. PPMGFP Sample Alf-AFP yes/no Vector Control 1 0.009 yes ExtracellularAlf-AFP 1 0.069 yes 2 0.154 yes 3 0.021 yes 4 0.003 no 5 0.001 no 60.019 no Cytoplasmic Alf-AFP 1 0.01 no 2 0.009 no 3 0.01 no 4 0.01 no 50.01 yes 6 0.01 yes 7 0.011 no 8 0.011 yes 9 0.011 yes 10  0.012 yes 11 0.01 no

The results from this experiment indicated that the extracellularytargeted Alf-AFP binary construct was capable of producing Alf-AFP intransgenic roots. A strong correlation between GFP positive roots andthose that expressed Alf-AFP was observed. However, the cytoplasmicallytargeted version of Alf-AFP did not accumulate Alf-AFP. Because thisconstruct was never tested, the expected result was uncertain. Thisexample demonstrates how rapidly constructs can be screened for geneexpression. Thus, one can quickly and cheaply screen for a geneticelement of interest using this method of generating transgenic hairyroots.

Example 2 Transformation of Potato

For generation of hairy roots on potato the same solutions are used asdescribed in Example 1. For plant material, potatoes that containnumerous branches are preferred. Potatoes do not need to be chilledprior to inoculation. Cut potato branches at nodes and place in Agroresuspension solution ( 1/10 strength B5 media plus 200 μMacetosyringone, 1 mM galacturonic acid, and 20 mM MES (pH5.4) to finalOD_(600nm)=0.3). Vacuum infiltrate and place in growth chamber at 22°C., 18 hr. light/6 hr. dark photo-period.

Production of Chimeric Potato Plants

Potatoes produce hairy roots much more rapidly than soybean. Roots willbegin to appear within two weeks. Adventitious roots generally do notappear. If they do, simply remove them with a scalpel. Co-transformationof hairy roots with a genetic element is between 70 and 90%. Up to 25independent hairy roots may form per stem.

The example below represents a study done on expression of ananti-fungal protein (AFP) from alfalfa (Alt). Two binary plasmids wereconstructed from pMON31873 (FIG. 1) using the Figwort Mosaic Virus (FMV)constitutive promoter, to drive expression of a cytoplasmically(pMON31892; FIG. 2) or extracellularly (pMON31896; FIG. 3) targetedAlf-AFP. Each construct was additionally linked to an enhanced 35Spromoter driving expression of the green fluorescence protein (GFP) andwas used to produce transgenic hairy-roots. Five weeks aftertransformation, transgenic roots were individually harvested, analyzedfor GFP expression by observing green fluorescence under a UV light andfrozen. Protein was extracted from root-tissues and used in astandardized ELISA assay using an antibody made specifically againstAlf-AFP to determine the amount of Alf-AFP present. Table 2 showsresults of the study.

TABLE 2 Expression of Alf-AFP and GFP in hairy roots of potato. PPM GFPSample Alf-AFP yes/no Vector Control 1 0.024 yes 2 0.017 yesExtracellular Alf-AFP 1 0.226 yes 2 0.02 no 3 0.023 yes 4 0.074 yes 50.024 yes 6 0.057 yes 7 0.016 no 8 0.044 yes Cytoplasmic Alf-AFP 1 0.022yes 2 0.019 yes 3 0.017 no 4 0.015 yes 5 0.012 yes 6 0.011 yes 7 0.007yes 8 0.004 yes

The results from this experiment indicated that the extracellularytargeted Alf-AFP binary construct was capable of producing Alf-AFP intransgenic roots. A strong correlation between GFP positive roots andthose that expressed Alf-AFP was observed. However, the cytoplasmicallytargeted version of Alf-AFP did not accumulate Alf-AFP. Because thisconstruct was never tested, the expected result was uncertain. Thisexample demonstrates how rapidly constructs can be screened for geneexpression. Thus, one can quickly and cheaply screen for a geneticelement using this method of generating transgenic hairy roots.

All publications and patent applications mentioned in this specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

Although the invention has been described in detail for the purpose ofillustration, it is understood that such detail is solely for thatpurpose, and variations can be made therein by those skilled in the artwithout departing from the spirit and scope of the invention which isdefined by the following claims.

1. A method for testing a genetic element for functionality in adicotyledonous plant, comprising the steps of: obtaining an explant froma dicotyledonous plant species wherein the hypocotyl explant has a cutend below the cotyledon; inoculating the explant with Agrobacteriumrhizogenes containing an exogenous genetic element capable of beingtransferred to the explant to create a transformed explant; culturingthe transformed explant in a manner to produce transgenic roots;transferring the transgenic roots to an environment to produce a stablechimeric plant; analyzing tissue from the transgenic roots for thefunction of the exogenous genetic element.
 2. The method of claim 1wherein the exogenous genetic element is a gene that confers resistanceto plant pathogens.
 3. The method of claim 1 wherein the exogenousgenetic element is a gene that confers an agronomic trait to the plant.4. The method of claim 1 wherein the exogenous genetic element is a genethat is involved in the enzymatic or metabolic activity of the plant. 5.The method of claim 1 wherein the exogenous genetic element is apromoter sequence.
 6. The method of claim 1 wherein the explant isselected from the group consisting of stem, hypocotyl or root tissue. 7.The method of claim 1 wherein the explant is a hypocotyl providing a cutend below the cotyledon.
 8. The method of claim 7 wherein the cut end ofthe hypocotyl is contacted with the Agrobacterium rhizogenes.
 9. Themethod of claim 8 wherein the Agrobacterium rhizogenes is strain K599.10. The method of claim 1 wherein the explant is obtained from adicotyledonous plant.
 11. The method of claim 10 wherein the plant issoybean, potato, or tomato.
 12. The method of claim 8 wherein transgenicroot development is initiated in the inoculated hypocotyl by placing theinoculated hypocotyl region in a media containing ¼ MS.
 13. The methodof claim 12 wherein the media further comprises a selectable agent. 14.The method of claim 13 wherein the selectable agent is kanamycin. 15.The method of claim 14 wherein the concentration of kanamycin in themedia is no more than about 50 mg/L.